学位论文详细信息
A NMR Investigation of Structure and Allostery in the Hsp70 Chaperone System.
NMR Spectroscopy of Large Proteins;Structural Biology;Hsp70 Chaperone Proteins;Residual Dipolar Couplings (RDCs);Paramagnetic Relaxation Enhancement (PREs);Protein Science;Biological Chemistry;Chemistry;Molecular;Cellular and Developmental Biology;Physics;Science (General);Science;Biophysics
Bhattacharya, AkashWalter, Nils G. ;
University of Michigan
关键词: NMR Spectroscopy of Large Proteins;    Structural Biology;    Hsp70 Chaperone Proteins;    Residual Dipolar Couplings (RDCs);    Paramagnetic Relaxation Enhancement (PREs);    Protein Science;    Biological Chemistry;    Chemistry;    Molecular;    Cellular and Developmental Biology;    Physics;    Science (General);    Science;    Biophysics;   
Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/75889/akashb_1.pdf?sequence=1&isAllowed=y
瑞士|英语
来源: The Illinois Digital Environment for Access to Learning and Scholarship
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【 摘 要 】

The Hsp70 Chaperone system is a complex molecular machine composed of proteins. It is responsible for assisting in protein folding/refolding/processing and transport. Hsp70 proteins have been historically associated with the ;;stress’ or ;;heatshock’ response of cells. They function in association with several co-chaperones in a delicately balanced and regulated two stage functional cycle. This system and its component molecules has been the subject of intensive efforts in structural biophysics for the last two decades. In this dissertation we present an analysis of the allosteric machinery, which operates in this molecular system. Using residual dipolar coupling analysis, a state-of-the-art method in solution nuclear magnetic resonance (NMR) spectroscopy, we have been able to detect distinct changes in the T.th-DnaK (the thermophilic Hsp70 protein) as the protein switches between the different stages of its ;;two-stroke’ functional cycle. Specifically, we have seen an opening of the nucleotide binding cleft in the ADP.PI state, without any exchange factor involved. Furthermore, we observed an opening of the IA/IIA interface cleft which would account for the linker being structured in the ATP state and explain how the interdomain allostery works in Hsp70s. Such changes have been observed for the first time in this field. We have also refined new methods to detect residual dipolar couplings and scrutinized our results with the most stringent possible statistical self-validation analysis.In the second stage of this dissertation, we have studied the interaction of E.coli DnaK (the bacterial Hsp70 molecule) with DnaJ (the equivalent bacterial co-chaperone) using paramagnetic relaxation enhancement in solution NMR. Based on this analysis, we propose a model for the interaction of DnaK with DnaJ, where DnaJ lies transversely across both domains of DnaK and might act as a ;;molecular crowbar’ in wedging the two domains apart. This study sheds new light on how the DnaJ co-chaperone system structurally interacts with the primary Hsp70 system and suggestsa possible mechanical model which explains how the allosteric machinery operates in the complex.

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